Synthetic antigenic peptide derived from Hepatitis B surface antigen

ABSTRACT

There is disclosed a new synthetic peptide which evokes an immunological response. The synthetic peptide, moreover, interacts with antibodies to Hepatitis B surface antigen (HBsAG). Thus, the synthetic peptide is useful as an immunizing agent in a vaccine as an active component thereof where it serves to produce antibodies in vivo which are protective against Hepatitis B virus. The synthetic peptide of the invention comprises the following sequence of amino acids: Arg Trp Met Met Leu Arg Arg(I) and preferably has the following sequence: Gly Tyr Arg Trp Met Met Leu Arg Arg Phe Gly (II).

This is a division of application Ser. No. 493,904, filed May 12, 1983.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a synthetic peptide, to a synthetic vaccinecomprising said peptide, to an agent for stimulation of antibodyproduction to a naturally occurring antigen, and to a process for theirpreparation.

2. Discussion of Prior Art

Hepatitis B virus (HBV) vaccines have been prepared from lipoproteinparticles containing hepatitis B surface antigen (HBsAg) isolated fromplasma of chronic HVB carries (McAuliffe et al., 1980). This source ofantigen is costly and scarce, therefore research was initiated toproduce the immunogen by recombinant DNA technology. Despite extensiveefforts, the yield of immunogenic material has been disappointing(Burrell et al., 1979; Edman et al., 1981, Mackay et al., 1981). Onlyrecently cloning the HBsAg gene led to production of lipoproteinparticles of full immunogenic potency in S. cerevisiae (Valenzuela etal., 1982). However, for practical purposes, the yield of HBsAgparticles was still very low. Therefore, several groups of workers haveattempted to develop a synthetic hepatitis B vaccine searching bycomputer the amino acid sequence of HBsAg for continuous antigenic sites(Atassi, 1980), either indirectly by exclusion of hydrophobic sequences(Vyas et al., 1981; Lerner et al., 1981; Dreesman et al., 1982) or bydirect search for hydrophilic structures (Hopp and Woods, 1981; Hopp,1981; Prince et al., 1982).

It has been reported that when the DNA for the HBsAg gene is split intoapproximately two equal halves and cloned in E. coli, it produces twoseparate antigenic peptides (MacKay et al., 1981). This information hasbeen confirmed by synthesis of antigenic (immunogenic) peptides derivedboth from the C-terminal (Lerner et al., 1981; Dreesman et al., 1982;Hopp and Woods, 1981; Hopp, 1981; Prince et al., 1982, Bhatnagar et al.,1982) and from the N-terminal (Lerner et al. 1981) halves of the HBsAgmolecule. When the above synthetic peptides were mapped on a diagram ofhydrophilicity of their amino acid residues, it was determined thatpractically all the peptides comprising the prominent hydrophilicdomains in the sequence of HBsAg have already been synthesized (resultsnot shown).

SUMMARY OF THE INVENTION

We have now discovered a new synthetic peptide which evokes animmunological response. The synthetic peptide, moreover, interacts withantibodies to hepatitis B surface antigen (HBsAg). Thus the syntheticpeptide is useful as an immunizing agent in a vaccine as an activecomponent thereof where it serves to produce antibodies in vivo whichare protective against Hepatitis B virus.

The synthetic peptide of the invention comprises the following sequenceof amino acids:

    Arg Trp Met Met Leu Arg Arg                                (I)

Preferably the synthetic peptide has the following sequence

    Gly Tyr Arg Trp Met Met Leu Arg Arg Phe Gly                (II)

Such preferred synthetic peptide includes the hydrophobic N-terminal andC-terminal residues (tyrosine and phenylalanine, respectively) as wellas a pair of terminal glycyl residues in the N-terminal and in theC-terminal positions. The glycyl residues are especially useful wherethe synthetic peptide is to be chemically connected to a carriersubstrate such as a physiologically compatible carrier. When disposed onsuch carriers, the synthetic peptide is useful as a vaccine for activeimmunization against hepatitis B virus.

The synthetic peptide of formulae I or II supra can be part of a largeror longer chain, including a longer amino acid sequence. It ispreferred, however that any amino acid chain containing this syntheticpeptide not exceed 50 amino acids. Preferably the amino acid sequencedoes not exceed 40 amino acids and still more preferably it does notexceed 30 amino acids. Very suitable are sequences containing less than20 amino acids, especially less than 15 amino acids. This heptapeptideof the invention (Formula I) or its extended undecapeptide (Formula II)can be disposed on a non-amino acid containing chain (e.g. polyethyleneglycol, PEG or its derivatives).

The synthetic vaccine is prepared either by chemically synthesizing achain of amino acids corresponding to the sequence of amino acids givensupra. The amino acid chain so obtained can thereafter, for use as avaccine, be disposed on a physiologically acceptable carrier, which canbe a polymer itself. The resultant composition can thereafter be dilutedwith a physiologically acceptable medium. The composition is then readyfor introduction into a host animal. The synthetic peptide can also beimmobilized on a soluble or insoluble substrate such as Sepharose whenit is intended to employ the same as a diagnostic agent.

It will be realized that the synthetic vaccine of the invention cancontain a single or a plurality of sequences given above and can bedisposed in an amino acid chain containing antigenic determinantscorresponding to other viruses or allergens. Similarly, the syntheticvaccine can contain a plurality of amino acid sequences of differentantigenic determinants of multiple known or unknown protein antigens orallergens. Thus the synthetic vaccine can contain a plurality of asingle antigenic determinant or can contain a single amino acid sequenceof a first antigen and another of a second antigen or allergen.

Where the synthetic peptide of the invention is part of a long chain,such as when there are more than one immunologically active amino acidsequence in the chain, the chain can contain residues of any of thefollowing moieties: segments of polyamino acid, polysaccharides,polyamides, vinyl polymers, ester polymers, polyacetals, polyolefins,polyphenyl sulfides, polycarbonates as well as bivalent organicradicals, including bivalent alkylene and other saturated or unsaturatedorganic compounds e.g. hydrocarbon radicals. These residues can havemolecular weights of up to 1,000,000, preferably between 10,000 and100,000, the molecular weight being determined by ultracentrifugation.

It will be realized that a chain containing the synthetic peptide cancontain a vaccine adjuvant. Such vaccine adjuvants include muramyldipeptide and its analogs which can be covalently bonded.

Alternatively, the vaccine can comprise a chain of amino acidscontaining one or more sequences of the synthetic peptide together withother chains of amino acids containing the same synthetic peptide ordifferent synthetic peptides corresponding to different antigens orallergens. These additional chains can be of the same or different chainlength. The chains which contain synthetic peptide sequences can beinterconnected with one another such as by crosslinking or by beingbonded directly thereto in the form of a branched chain, or therespective chains are bonded to a central "carrier". It is contemplatedthat the vaccine contain a plurality of the same or different syntheticpeptide sequences of the invention. In particular, it is contemplatedthat a vaccine contain between 1 and 1,000 of such sequences, percovalent unit. It can also have present in addition thereto between 1and 1,000 peptide sequences per covalent unit of a different antigen orallergen or plurality of different antigens or allergens, all asdesired.

The synthetic peptide of the invention requires proper presentation inorder to elicit an immune response. To this end, a carrier is providedtherefor. The "carrier" is simply a physiologically acceptable mass towhich the synthetic peptide is attached. A carrier can comprise a chainof amino acids or other moieties and to that end it is specificallycontemplated to use as a carrier or dimer, oligomer, or higher molecularweight polymer of sequences containing amino acids of the syntheticpeptide. Thus the synthetic peptides can be polymerized to build up achain of two or more repeating units so that repeating sequences serveboth as "carrier" and immunologically active site. Stated differently,an independent carrier is not required. It is preferred that alternativecarriers comprise some substance, animal, vegetable, or mineral, whichis physiologically acceptable and functions to present the syntheticpeptide so that it is recognized by the immune system of a host andstimulates a satisfactory immunological response. Thus, a wide varietyof carriers are contemplated, and these include materials which areinert, which have biological activity and/or promote an immunologicalresponse. For instance, proteins can be used as carriers and there isincluded within such subclass, human serum proteins, tetanus toxoid,etc.

Polysaccharides are also contemplated as carriers, and these includeespecially those of molecular weight 10,000 to 1,000,000 including inparticular starches, dextran, agarose, ficoll or its carboxy methylderivatives and carboxy methyl cellulose.

Polyamino acids are also contemplated for use as carriers and thesepolyamino acids include, among others, polylysine, polyalanylpolylysine, polyglutamic acid, polyaspartic acid and poly (C₂ -C₁₀)amino acids.

Vaccines can be used as carriers for the synthetic peptide provided bythe invention. In other words, the synthetic peptide residues providedby the invention can themselves be attached to other vaccines includingvaccines for measles, influenza, smallpox, polio, diphtheria,pneumonococci, meningococci, and the like.

Organic polymers can be used as carriers, and these polymers includepolymers and copolymers of amines, amides, olefins, vinyls, esters,acetal, polyamides, carbonates, ethers, phenylene sulfides, silicones,urea formaldehyde condensation products, phenol formaldehydecondensation products, urethanes, melamine formaldehydes, epoxy resins,acrylic resins, allyl resins, and the like. Generally speaking, themolecular weight of these polymers will vary dramatically. The polymerscan have from two repeating units up to several thousands e.g., twothousand repeating units. Of course, the number of repeating units willbe consistent with the use of the vaccine in a host animal. Generallyspeaking, such polymers will have a lower molecular weight, say between10,000 and 100,000, determined in accordance with the procedure setforth above.

Inorganic polymers can also be employed. These inorganic polymers can beinorganic polymers containing organic moieties. In particular, silicatescan be used as carriers. It is preferred that the carrier be one whichis an immunological adjuvant. In such cases, it is particularlycontemplated that the adjuvant be any one of the following: muramyldipeptide or its analogs.

The carrier can also be the residue of a crosslinking agent employed tointerconnect a plurality of peptides containing chains. The crosslinkingagent can be one which interconnects the chains at a point containingthe sequence of the synthetic peptide amino acids. Alternatively, thecrosslinking agent can interconnect a plurality of chains at a pointother than where the synthetic peptide is located. Crosslinking agentswhich are contemplated include crosslinking agents which have as theirfunctional group an aldehyde, carboxyl, amine, amido, imido orazidophenyl, group. In particular, there is contemplated the use ofglutaraldehyde as a crosslinking agent, a divalent imido ester or acarbodiimide. Particularly contemplated divalent imido esters are thoseof the formula ##STR1## wherein m is 1 to 13 and R is an alkyl group of1 to 4 carbon atoms.

Particularly contemplated carbodiimides for use as crosslinking agentsinclude cyclohexylcarbodiimide, ethyldimethylaminopropyl carbodiimide,N-ethylmorpholino cyclohexyl carbodiimide, diisopropyl carbodiimide,etc.

It should be understood that the vaccine of the invention can be inadmixture with other proteins and these proteins include the proteins ofknown antigens or allergens. Thus, when it is stated herein that thevaccine is characterized by the absence of an amino acid sequencecorresponding to the entire protein antigen or allergen it is meant thatnotwithstanding the absence of the amino acid sequence of the entireprotein antigen or allergen, the composition functions as a vaccine,i.e. provides protective immunization by formation of antibodies in thecase of an antigen or a lessening of allergic sensitivity in the case ofan allergen.

In addition to the carriers named above, or as an alternative thereto wecan use a carrier comprising a straight or branched substituted orunsubstituted, saturated or unsaturated hydrocarbon residue of at leasttwelve carbon atoms as disclosed in copending application Ser. No.358,150 of Thomas P. Hopp, assigned to the assignee hereof, thedisclosure of which is hereby incorporated hereby by reference. Asdisclosed therein, in particular, the carrier is one having at leasttwelve carbon atoms in a chain whose chain is either an alkyl or alkenylgroup. Such alkyl or alkenyl group can have up to 36 carbon atoms but ispreferably in the range of C₁₂ to C₂₄. These hydrocarbon residues can beprovided by fatty acids by simple coupling of the fatty acid moiety to aterminal functional group of the synthetic peptide by relatively routinechemistry. There is contemplated, however, carrying the syntheticresidue on such hydrocarbon residues without the use of the carboxylicacid functional group of the fatty acid whereby the synthetic peptide isjoined to the hydrocarbon residue without a carbonyl connecting link.

Thus, our invention can be described broadly as a composition comprisinga synthetic peptide of the sequence given supra in combination with aspecific carrier as represented by the formula ##STR2## wherein m is 0or 1:

R is a substituted or unsubstituted alkyl or alkenyl radical of at least12 carbon atoms; and

Peptide is the residue of formulae I

or II, supra.

Referring to the formula above we can couple the synthetic peptidemoiety to an alkyl or alkenyl group of at least 12 carbon atoms byblocking all those amino groups of the synthetic peptide residue so thatthey are free of reactivity to a carboxylic acid except that a terminalamino group remains available for reaction. The terminal amino group ofthe synthetic peptide then reacts with a moiety which supplies acarboxylic acid group whereby condensation of a hydrogen atom of theamino group with the hydroxyl group of carboxylic acid group(dehydration) effects interlinkage of the synthetic peptide with thecarboxylic acid group in accordance with the following equation:##STR3##

In accordance with this reaction there is formed a composition asdefined in equation I above wherein m=1. also envisaged, however, indisposing these synthetic peptides on a C₁₂ -C₃₆ alkyl or alkenyl moietywithout using a carboxylic acid or similar functional group to link withthe terminal amino group. Thus, for instance, there is contemplated asubstitution reaction in accordance with the following equation:##STR4## in which case there is formed a synthetic vaccine withinformula I above wherein m is 0. Numerous alternative routes to disposinga synthetic peptide on a C₁₂ to C₃₆ alkyl or alkenyl group are apparent;these invariably linking the synthetic peptide to the alkyl or alkenylmoieties via a terminal amino group of the synthetic peptide moiety.

In forming a synthetic vaccine in accordance with this invention, it ispreferred to use a fatty acid of C₁₂ to C₂₄. Particularly contemplatedfatty acids include the following:

Palmitic

Stearic

Behenic

Oleic

The synthetic peptide can be prepared using the Merrifield solid phaseprocedure to build up the appropriate sequence of L-amino acids from thecarboxyl terminal amino acid to the amino terminal amino acid. Startingwith the appropriate carboxyl terminal amino acid attached to apolystyrene or other appropriate resin via chemical linkage to achloromethyl group, benzhydrylamine group, or other reactive group ofthe resin, amino acids are added one by one using the followingprocedure for each:

(a) peptidyl resin is washed with methylene chloride;

(b) neutralized by mixing for 10 min. at room temperature with 5% (v/v)diisopropylethylamine (or other hindered base) in methylene chloride;

(c) washed with methylene chloride;

(d) an amount of amino acid equal to six times the molar amount of thegrowing peptide chain is activated by combining it with one-half as manymoles of a carbodiimide (e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, etc.) for 10 minutes at 0° C., to form thesymmetric anhydride of the amino acid. The amino acid used should beprovided originally as the N-α-butyloxycarbonyl derivative, with sidechains protected with benzyl esters (aspartic and glutamic acids) benzylethers (serine, threonine, cysteine, tyrosine), benzyloxycarbonyl groups(lysine) or other protecting groups commonly used in peptide synthesis;

(e) the activated amino acid is reacted with the peptidyl resin for 2hours at room temperature, resulting in addition of the new amino acidto the end of the growing peptide chain;

(f) the resin is washed with methylene chloride;

(g) the N-α-butyloxycarbonyl group is removed from the most recentlyadded amino acid by reacting with 30% (v/v) trifluoroacetic acid inmethylene chloride for 30 minutes at room temperature.

(h) the resin is washed with methylene chloride;

(i) steps (a) through (h) are repeated until the required peptidesequence has been constructed.

The peptide is then removed from the resin and simultaneously theside-chain protecting groups are removed, by reacting with anhydroushydrofluoric acid containing 10% (v/v) of anisole. Subsequently, thepeptide can be purified by gel filtration, ion exchange or high pressureliquid chromatography, or other suitable means.

In some cases, chemical synthesis can be carried out without the solidphase resin, or on soluble carriers e.g. polyethylene glycol orderivatives thereof, in which case the synthetic reactions are performedentirely in solution. The reactions and the final product are otherwiseessentially identical.

The Merrifield solid phase synthesis for synthetic peptides is aparticularly desirable approach to formation of a fatty acid carriedsynthetic peptide, since it provides a convenient means for attachmentof the carrier in accordance with the invention, although it should beunderstood that liquid phase approaches can also be employed. TheMerrifield solid phase approach involves connecting amino acids to oneanother where the pendent reactive groups, e.g., amino, hydroxyl,carboxyl, imidazol groups, are blocked. After the final amino acid hasbeen coupled, the N-terminus is deblocked and a fatty acid or othersuitable large lipophilic substituent or component supplying a C₁₂ toC₃₆ alkyl or alkenyl group is reacted by procedures outlined above foruse in amino acid couplings, the procedure is carbodiimide mediatedpeptide (amide) bond formation, hydroxybenzotriazole ester addition oraddition of a fatty acid symmetrical or asymmetrical anhydride.

This results in a peptide with covalent N-terminal fatty acid or similarmoiety. The peptide is then removed from the resin by typicalhydrofluoric acid treatment, and purified if necessary.

Analogously, one can form chains containing a plurality of the syntheticpeptides of the invention or with amino acid sequences corresponding todifferent antigens or allergens by the following technique: An aqueoussolution of the synthetic peptide or peptides is mixed with awater-soluble carbodiimide (e.g. ethyldimethylaminopropylcarbodiimide).This results in polymerization of the peptide(s); depending on the useof the side chain blocking groups mentioned above, either straight chainor branched polymers of the synthetic peptide can be made.

If desired, the synthetic peptide containing chain employed in thevaccine of the invention can have bonded thereto a chain of any of thefollowing moieties: polypeptide, polyaminoacid, polysaccharide,polyamide, polyacrylamide which can serve as a stabilizing chain or as abridge between synthetic peptide sequences. Such chains are availablecommercially or, in the case of polyamino acids, are formed by a processwhich comprises: mixing a solution of epitope synthetic peptide with asolution of the N-carboxylanhydride of the amino acid and allowing abase-catalyzed polymerization to occur, which is initiated by the aminegroups of the peptide.

The disposition of a chain or chains on a "carrier" is effected asfollows:

1. Protein Carrier. The protein and the synthetic peptides are dissolvedtogether in water or other suitable solvent, and covalently linked viaamide bonds formed through the action of a carbodiimide. The resultingproduct may contain one or more copies of the peptide per proteinmonomer.

2. Polysaccharide Carriers: Oligosaccharide carriers should havemolecular weights in the range of 1,000 to 1,000,000. In order tocovalently link these to H-epitope peptides, suitable functional groupsmust first be attached to them. Carboxyl groups may be introduced byreacting with iodoacetic acid to yield carboxymethylatedpolysaccharides, or by reacting with carbonyldiimidazole to yieldactivated carbonyl esters. Carboxymethyl polysaccharides are coupled tothe peptide by a carbodiimide reaction, while the activated carbonylesters react spontaneously with peptides. Multiple copies of thesynthetic peptide should be attached to each oligosaccharide unit.

3. Polyamino Acid Carriers. These carries should have molecular weightsin the range 1,000 to 1,000,000. Polylysine and polyornithine haveprimary amino groups on their side chains; polyaspartic acid andpolyglutamic acid have carboxyl groups. Peptides may be coupled to thesevia amide bonds using the carbodiimide reaction. Another carrier thatprovides amino groups for coupling is polylysine to which polyalaninehas been attached to the side chains of the lysine residues. Thesynthetic peptide may be attached to the ends of the polyalanine chains,also by a carbodiimide reaction. Multiple copies of the syntheticpeptide should be attached to each oligopeptide unit.

The respective synthetic peptide containing chains can be linked to oneanother by a cross-linking agent. Generally speaking, the cross-linkingagent can be any one of a type identified above. Crosslinking iseffected by reacting the synthetic peptide containing residue with thecrosslinking agent as follows:

Reaction with glutaraldehyde or a bis-imidate (e.g.dimethylsuberimidate) in aqueous solution results in polymerization ofthe synthetic peptide containing moiety with the cross-linking reagentforming covalent bridges between peptide monomers.

By the procedure of the invention there is realized a vaccine which canbe characterized by the absence of an amino acid sequence of the entireprotein antigen or allergen. The hepatitis B vaccine can be synthesizedfree of other peptide sequences of the hepatitis B surface antigenprotein, or other proteins found in the virion. Vaccines can besynthesized which are free of biologically produced components, free ofviral components whether they be active or inactive, free of antibodies,free of deoxyribonucleic acid (DNA) and free of lipids, and are,therefore, likely to be substantially free from undesirable side effectscommonly found with other vaccines (unintentional infection with virus,allergic reactions, fevers, etc.).

The synthetic vaccines are characterized by exceptional specificity andevoke an unusual and special response when introduced into a hostanimal. Whereas a vaccine made of natural material and introduced into ahost animal usually evokes an immunological response by the reaction ofantibodies specific to a number of distinct epitopes present on theantigens found in that vaccine, when the vaccine of the presentinvention is introduced into a host animal, it causes the formation ofantibodies which are mono-specific, i.e., are specific to the singleantigenic site on the vaccine. The synthetic peptide produced not onlyantibodies to a single specific antigenic determinant on the naturalvirus but also produce a mixture of antibody molecules, all of which arespecific to that same antigenic determinant. Hence, the antibodies ofthe invention are "hetero-molecular". This provides different moleculesall of which are suited to bind the natural virus or allergen. Some ofthese molecules due to their size, shape, mobility andhydrophilic/hydrophobic properties may act faster or more strongly thanothers. This insures broad and specific protection.

It is to be realized, moreover, that the antibodies are "mono-specific"in a different sense than are antibodies produced from compositionscontaining the entire or virtually the entire amino acid sequence of thenatural viral protein. The latter produce antibodies to a plurality ofdifferent antigenic determinants of the same virus but not to otherviruses. Thus, while being "mono-specific" in the sense of producingantibodies only to that virus they produce antibodies which are notspecific to a single antigen determinant of the virus.

Thus the synthetic peptide of the invention can be used to form immuneglobulins comprising the single antigenic determinant specificheteromolecular antibodies of the invention. These antibodies can beproduced in animals such as chimpanzees. They serve as diagnostic agentswhere due to their heteromolecular but single determinant specificcomposition they insure a response to naturally occurring virus in atest serum. Thus they can be used in serological testing to identify thepresence of Hepatitis B virus.

In the preparation of a vaccine the concentration of the same in thephysiologically acceptable medium will vary depending on the number ofsynthetic peptides or other residues therein. Generally speaking, theactive component of the vaccine can be present in a concentration whichis lower than the concentration of active material in known vaccinessince in the known vaccines, higher concentrations were required inorder to have present the required number of antigenic determinants toevoke the desired immunological response. The vaccine concentrationwill, of course, vary from vaccine to vaccine. Generally speaking, itsconcentration will be from 5 to 1,000 μg, preferably 20 to 50 μg perdose to give suitable immunogenicity. It is particularly contemplated touse the vaccine in a dosage of 1 to 100 μg., especially 1 to 20 μg. perdose.

The HBsAg vaccine will have sufficient potency to provide an anti-HB_(s)titer of at 100 milli-international units (MIU) in comparison to the WHOInternational HBIG Standard in at least four chimpanzees immunized with2 doses of the standard vaccine in accordance with the recommendedschedule, the anti-HB_(s) remaining detectable at a titer of greaterthan 10 mIU for at least a year following the onset of immunization ofthe chimpanzees. Naturally, the vaccine concentration can vary fromthese concentrations in accordance with the desired effect.

The vaccine can be administered by subcutaneous or intramuscularinjection. While the preferred route depends upon the particularvaccine, intramuscular injection is generally suitable. Frequency ofadministration depends upon the vaccine and the number and type ofsynthetic peptide moieties and their concentration in the activecomponent. Generally speaking the vaccine is administered in two dosesabout one month apart followed by a booster at six months to one yearafter primary immunization. Of course, the dosage depends upon the sizeof the host animal being inoculated. The subsequent doses or the boosterdepends on the level of antibody in the blood as a result of the initialimmunization. Licensable adjuvants conventionally employed in vaccinemanufacture can be utilized.

In the case of the hepatitis vaccine of the invention, the same isrecommended for all persons at risk of developing hepatitis B infectionand particularly those at especially high risk such as patients andstaff on hemodialysis unit, medical personnel, persons of tropicalpopulations and those visiting the tropics. In the case of tropicalpopulations, particularly in Africa, Asia, the Medeterranean region andSouth America, where high incidence of hepatitis B infections has beenconsistently observed, the vaccine should be administered sufficientlyearly in life to prevent acquisition of chronic carrier state infectionwhich tend to occur in these regions within the first five years oflife. In fact, the vaccine is useful for all persons not alreadyprotected against hepatitis B infections as a result of prior immunity.

BRIEF DESCRIPTION OF DRAWINGS

Referring to the annexed drawings, all of which are graphs plotting onefunction or observation against another:

FIG. 1A is a time course diagram of anti-HBs response in rabbitsinjected with the peptide conjugated to tetanus toxoid as discussedbelow.

FIG. 1B is the anti-peptide antibody profile of sera collected fromrabbits inoculated with the peptide conjugated to tetanus toxoid.

In order to more fully illustrate the invention and the manner ofpracticing the same, the following examples are presented.

EXAMPLES Chemicals and Reagents

Chemicals were of analytical grade or otherwide specified. The syntheticpolypeptide carrier Poly-D,L-Alanyl-Poly-L-Lysine (A,L: M.W. 60,000) waspurchased from Miles Laboratories (Elkhart, Ind.). Tetanus toxoid (LotNo. CPTxd-35) was a gift from Dr. George Grady (Biological Laboratories,The Commonwealth of Massachusetts). The monoclonal antibodies wereacquired from Dr. Courouce of the National Blood Transfusion Center,Paris.

Peptide Synthesis

The peptide was synthesized in a liquid phase using as a carrierpolyethylene glycol 5000 (PEG) derivatized with p-hydroxymethyl-benzoicacid (Bayer and Mutter, 1974). The peptide was released from theC-terminal esteric bond with PEG by saponification and deprotectedaccording to a published procedure (Kito et al., 1980).

Peptide Analysis

The purified peptides were analyzed by TLC on silica gel plates with twosolvent systems composed of BuOH-Pyridine-AcOH-H₂ O mixed in a ratio(v/v) of 5:5:1:4 and 30:20:6:24 respectively. The amino acid compositionwas determined after acid hydrolysis (6N HCl, 110° C., 24 hrs.) in aBeckman Model 119 Amino Acid Analyzer.

Coupling of Purified Peptides to Carriers by Carbodiimide

The original PEG-peptide and the free peptide (released from PEG) wascoupled either to tetanus toxoid (TT) or to a synthetic polypeptidecarrier (A,L) with carbodiimide in the following molar ratios:TT-peptide-PEG 1:50; TT-peptide 1:50; and A,L-peptide 1:2, according topublished procedures (Bauminger and Wilchek, 1980; Goodfriend et al.,1964, Nash et al. 1980).

Immunization of Animals

Three groups of Balb/c mice (10 in each) obtained from JacksonLaboratory (Bar Harbor, Maine) were injected subcutaneously on day 1with 25 μg of the original PEG-peptide coupled to tetanus toxoidhereinafter (TT-peptide-PEG) or with 5 μg of the free peptide coupled sotetanus toxoid (TT-peptide) or the peptide synthetic carrier conjugate,(A, L-peptide) in 0.1 ml of 0.15M NaCl, 0.01M Phosphate pH7.2(PBS) mixedwith an equal volume of Freund's complete adjuvant (CFA). The mice wereboosted with the same amount of conjugates suspended in incompleteFreund's adjuvant (IFA) 30 days after the primary inoculation. Theanimals were bled from the tail vein 30 and 60 days respectively afterthe primary inoculation.

Two white New Zealand rabbits were injected subcutaneously with 50 μg ofpeptide (based on the free peptide) conjugated to tetanus toxoid in 0.25ml PBS mixed with an equal volume of CFA on day 1 and boosted with thesame amount of sample mixed with IFA 30 days after the primaryinoculation. The rabbits were bled weekly from the marginal ear vein.

IMMUNOLOGICAL ASSAYS Competition RIA for a Peptide Conjugate

The antigenic activity of the peptide-synthetic carrier conjugate(A,L-peptide) was determined by competition RIA using ¹²⁵ I-HBsAg asfollows:

The wells in a microtitration plate were coated directly by incubationwith 50 μl of supernatant media (diluted 1:10 in PBS) from each of thehybridoma cell cultures for 2 hours at 37° C. and overnight at 4° C.After recovering the antibody the wells were washed 3× with PBS andincubated for additional four hours at 37° C. with 100 μl of 1% bovineserum albumin (BSA) in PBS (containing 10% Ausab negative controlserum). The wells were washing again with PBS and used directly for theassay as follows: A 50 μl volume of the conjugate (100 μg/ml; based onthe peptide moiety) suspended in negative control serum was mixed with50 μl of ¹²⁵ I-HBsAg (Ausab reagent diluted 1:10 in negative controlserum) in an antibody coated dilution plate. After incubation at roomtemperature overnight, the plates were washed and the individual wellscut out and counted in a gamma counter. The results were expressed aspercent of reduction of cpm in the presence of the conjugated peptideagainst the total cpm in the controls.

Determination of anti-HBs by RIA

The anti-HBs titers of sera were determined under standard conditionsusing the AUSAB reagents (Abbott Laboratories). Aliquots of 2×0.1 ml ofsera (diluted with an equal volume of negative control sera) and 2×0.2ml of rabbit sera were used for the assay. The titers were expressedeither as ratio units of cpm of samples to cpm of negative controls(P/N) or the results were quantitated in comparison to the WHOInternational HBIG Standard and expressed as milli-international units(mIU) according to a published procedure (Wainfan, 1976).

Determination of Anti-Peptide Titers by ELISA

The anti-peptide antibody titers were determined by ELISA usingcommercial reagents (New England Nuclear and DAKO Corp.) The wells in amicrotitration dilution plate were coated with 100 μl of the syntheticpeptide (peptide-PEG) at a concentration of 100 μg/ml and 50 μl ofserial 2-fold dilutions of sera were applied. Antibody binding wasdetected as described by Bittle et al (1982). The plates were read 492nm in a MICROELISA MRJ80 (DYNATECH Corp.) automatic plate reader and thevalues presented are expressed as reciprocals of the serum dilutionshowing at least 50% maximal O.D. reading.

RESULTS

The purified original PEG-peptide and the free peptide (released fromPEG) were homogeneous when tested by TLC in two different solventsystems (see Methods). The amino acid composition of the peptidedetermined after acid hydrolysis (not corrected for decomposition) wasthe following: Leu: 1.0(1); Phe: 1.0(1); Gly: 1.7(2); Met: 1.6 (2); Tyr:0.9 (1); Arg: 2.8 (3); Trp; not determined (should be 1). The numbers inparentheses represent the theoretical number of residues. These resultsare in agreement with the expected amino acid sequence:Gly-Tyr-Arg-Trp-Met-Met-Leu-Arg-Arg-Phe-Gly.

The results of immunization summarized in Table 1 show a rather weakslowly increasing anti-HBs response in mice injected with the originalPEG peptide coupled to tetanus toxoid (TT-peptide-PEG). When the freepeptide (released from PEG) coupled to tetanus toxoid (TT-peptide), orto the synthetic carrier (A,L-peptide) were injected into mice, bothconjugates showed a fairly strong immune response (eight of ten miceresponded). There was an approximately two-fold increase in titers afterthe second injection indicating a relatively weak boosting effect.

When the peptide-tetanus toxoid conjugate was injected into two rabbitsboth elicited a fairly strong anti-HBs response (FIG. 1A). The same seraanalyzed for anti-peptide titers by ELISA (Bittle et al., 1982) showed apattern similar to anti-HBs (FIG. 1B).

The ability of the conjugates to bind with monoclonal antibodies wastested by a microtitration plate RIA in competition with ¹²⁵ I-labeledpurified HBsAg. A total of 19 different monoclonal antibodies were usedbut only five coated adequately. The peptide-synthetic carrier conjugateshowed a positive reaction (>50% inhibition) in plates coated with 4 of5 of the antibodies which could be tested (Table 2). These resultsindicate that the peptide carries an antigenic determinant whichinteracts with monoclonal antibodies produced against the components ofthe naturally occurring virus. Thus the purely synthetic peptide whoseamino acid sequence is a derivative of a short sequence of the naturalantigen is immunologically active with such naturally producedantibodies.

                  TABLE 1                                                         ______________________________________                                        ANTI-HBs TITERS IN MICE IMMUNIZED WITH PEPTIDE-                               CARRIER CONJUGATES                                                                       Amount per   Range of                                              Immunizing Mouse        Antibody  Titers (P/N)*                               Preparation                                                                              (μg)      Day 30    Day 60                                      ______________________________________                                        TT-peptide-PEG                                                                           25           2.1-4.8 (4)                                                                             2.5-7.7 (7)                                 TT-peptide 5            2.9-16.2 (8)                                                                            2.6-33.2 (8)                                A,L-peptide                                                                              5            3.0-8.7 (8)                                                                             3.3-17.7 (8)                                ______________________________________                                         *Only positive titers are listed. Numbers in parenthesis represent number     of mice responding out of the ten mice in each group.                    

                  TABLE 2                                                         ______________________________________                                        MICROTITRATION PLATE COMPETITION RIA FOR A,L-                                 SYNTHETIC-PEPTIDE USING MONOCLONAL -ANTI-HBs ANTIBODIES                                              Antibody*                                                       Monoclonal    Titers    Percent                                      Number   Antibodies    (P/N)     Inhibition                                   ______________________________________                                        1        16-A8         16.0      69.3-70.6                                    2         20-A20       54.7      57.3-62.6                                    3         20-A92       49.6      51.2-60.1                                    4        107-A3        19.2       6.6-18.5                                    5        112-A51       16.4      59.1-79.4                                    ______________________________________                                         *The amount of antibody coating the wells was determined under the same       conditions as in the samples except for using 50 μl of the .sup.125        IHBsAg reagent diluted directly with 50 μl of negative serum (see          Methods).                                                                

    ______________________________________                                        GLOSSARY                                                                      Amino Acid          Abbreviation                                              ______________________________________                                        Arginine            Arg                                                       Glycine             Gly                                                       Methionine          Met                                                       Isoleucine          Ile                                                       Leucine             Leu                                                       Tyrosine            Tyr                                                       Phenylalanine       Phe                                                       Tryptophan          Trp                                                       ______________________________________                                    

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What is claimed is:
 1. A synthetic peptide consisting essentially of thefollowing sequence of amino acids

    Arg Trp Met Met Leu Arg Arg                                (I)

wherein said peptide is disposed on a carrier.
 2. A synthetic peptideaccording to claim 1, wherein said carrier is a physiologicallyacceptable carrier.
 3. A synthetic peptide according to claim 2, whereinsaid carrier comprises tetanus toxoid.
 4. A synthetic peptide accordingto claim 1, wherein said carrier comprises polyethylene glycol.